Abstract
BACKGROUND AND AIM: The Ovar-DRB1 gene, a key component of the sheep main histocompatibility complex (MHC) class II region, plays a critical role in antigen presentation and immune responsiveness. Despite the well-documented hypervariability of exon 2 in many sheep breeds, no study has yet examined DRB1 allelic composition in Edilbay sheep, a Kazakh breed highly adapted to harsh continental steppe conditions. This study aimed to characterize the diversity of Ovar-DRB1 exon 2 alleles in Edilbay sheep and to identify novel allelic variants using Sanger sequencing. MATERIALS AND METHODS: Blood samples from 50 Edilbay sheep reared at a breeding farm in Kazakhstan were subjected to DNA extraction and DRB1 exon 2 amplification using validated primers. Polymerase chain reaction products were purified and sequenced using Sanger sequencing. Allele identification was performed through pairwise sequence alignment in SnapGene and reference comparison with the Immuno Polymorphism Database of Major Histocompatibility Complex (IPD-MHC) database. Ambiguous chromatograms and overlapping nucleotide peaks were assessed for potential novel allelic patterns. Genetic diversity indices (Ho, He, Ne, and Shannon's H') were calculated. RESULTS: A total of 25 known DRB1 alleles were identified in the Edilbay sheep population. Genetic diversity parameters demonstrated extremely high immunogenetic variation, with observed heterozygosity (Ho) of 0.94, expected heterozygosity (He) of 0.90, an effective number of alleles (Ne) of 16.7, and Shannon's index (H') of 3. Several chromatograms showed overlapping peaks or substitution patterns inconsistent with known alleles, including variations at positions 243-244 and multiple additional polymorphic sites. These patterns indicate the presence of putative novel alleles that could not be unambiguously assigned by direct Sanger sequencing. Approximately 20% of samples contained undocumented variants or low-quality chromatograms requiring further resolution. CONCLUSION: This study presents the first comprehensive molecular characterization of Ovar-DRB1 exon 2 in Edilbay sheep, revealing exceptionally high genetic diversity and strong evidence for previously undescribed alleles. These findings broaden the catalog of DRB1 variants and highlight the breed's adaptive immunogenetic potential. Further investigations using allele-specific amplification, cloning, or next-generation sequencing are recommended to precisely identify novel variants and explore associations with disease resistance and environmental adaptation.